The Thalassemia Syndromes: New Insights

Abstract

Thalassemia is characterized by impaired synthesis of globin chains in hemoglobin. Supportive care for this condition includes regular transfusions and adequate iron chelation. Hemopoietic stem cell transplant (HSCT) is the only curative option available at present to most of the patients. The currently accepted indication for allogenic HSCT is transfusion dependency. For patients with available HLA-matched siblings or related or unrelated donors, a transplant should be offered as soon as possible to avoid transfusion-associated complications. The three risk factors are the presence of hepatomegaly >2 cm, the presence of liver/portal fibrosis and a history of inadequate chelation. Stem cells for HSCT can be obtained from bone marrow, peripheral blood and cord blood. In the majority, all the transplant centres across the world use bone marrow as a stem cell source as it is associated with a lesser incidence of GVHD (especially chronic) as compared to peripheral blood because of the high concentration of T lymphocytes in the latter. Conditioning regimen is being evolved from myeloablative to reduced intensity conditioning to reduced toxicity myeloablative conditioning regimens. Post-transplant management includes monitoring of engraftment and chimerism. It also aims at infection prophylaxis, prevention of GVHD, hematopoietic support and management of iron overload post-transplant.

1. Introduction

Thalassemia is an inherited blood disorder characterized by impaired synthesis of globin chains in hemoglobin. It is a recessive monogenic disorder and 5% of population globally are carrier of this disease [1]. The estimated global prevalence of severe beta thalassemia is 288,000 per annum [2]. Transfusion-dependent thalassemia is associated with severe anemia and its complications, iron overload affecting multiple organs due to frequent transfusions, extramedullary hematopoiesis and hepatosplenomegaly. Supportive care for this condition includes regular transfusions and adequate iron chelation. However, hemopoietic stem cell transplant (HSCT) is the only curative option available at present [2].

Allogenic stem cell transplant in thalassemia as a cure was first reported in 1982 from a human leukocyte antigen (HLA) identical sibling donor [3]. Following this, more than 3000 successful transplants in thalassemia have been done [4]. In the 1980s and early 1990s, the Pesaro group in Italy pioneered the therapeutic approach to transplant in thalassemia [5, 6, 7, 8, 9, 10, 11]. This was later accepted worldwide. Since then, there has been gradual improvement in outcomes over the past 20 years due to improved strategies of conditioning, risk stratification and better control of complications. Overall survival (OS) and Thalassemia free survival (TFS) rates of thalassemia have improved to around 90% and 80%, respectively [12, 13]. The best available donor for HSCT is a full HLA-matched sibling donor (MSD) or family member. However, such donors are not easily available and hence strategies are coming up to use matched unrelated and mismatched donors as a source of stem cells for transplant. Till now their outcomes are inferior to matched related donors [14, 15]. Hence case to case basis discussions among transplant physicians and parents are required to choose between transplant or supportive therapies as part of the management of the disease when matched related donors are not available.

2. Indication of transplant and risk stratification

The currently accepted indication for allogenic HSCT is transfusion dependency. For patients with available HLA-matched siblings or related or unrelated donors, a transplant should be offered as soon as possible to avoid transfusion-associated complications [16]. HSCT is not chosen for patients with severe organ damage e.g. uncompensated cirrhosis.

In the early 1990s, the Pesaro group identified three patient-related risk factors which can affect the outcomes of transplants. These three risk factors were incorporated to stratify three classes of patients with the best outcomes available from class1 and the worst from class 3 [11].

The three risk factors were presence of hepatomegaly >2 cm, the presence of liver/portal fibrosis and a history of inadequate chelation. The quality of chelation was characterized as regular when deferoxamine therapy was initiated no later than 18 months after the first transfusion and was administered subcutaneously for 8–10 h continuously for at least 5 days/week. Any deviation from this regimen was defined as irregular chelation.

Class 1 had no risk factors, class 2 had one or two risk factors, and class 3 had three risk factors. The 3-year overall survival (OS) for class 1 was 94% and dropped to 61% for class 3. Class 3 also contained a group of very high-risk (HR) patients, typically aged ≥7 years and with liver size ≥5 cm from the costal arch.

Age older than 14 years is an independent risk factor. If MSD HSCT is performed before 14 years of age, procedure-related mortality is <10%. This decreases to <5% when performed before 5 years of age [17].

3. Pretransplant evaluation

In addition to classical pre-HSCT evaluation, the following tests need to be done:

Liver iron concentration: Assessment of liver Iron is done by liver biopsy or liver MRI, this can be avoided in patients with age less than 3 years. Compensated Cirrhosis (e.g. Child-Pugh class A) can have an impact on the prognosis and outcome of the transplant. It is not a contraindication to transplant but it should be weighed as a factor to choose HSCT on a case-to-case basis. Liver histology—with particular attention to the degree of fibrosis (for this purpose liver biopsy remains the preferred tool, over liver elastography) should be done. For the evaluation of liver fibrosis by biopsy, Knodell’s numerical scoring system should be used [18].

Viral hepatitis tests: Viral hepatitis is not a contraindication to HSCT but tests should be done and appropriate antiviral drugs should be started and viral load should be lowered before starting of transplant.

Cardiac assessment: Electrocardiography or Echocardiography are appropriate tests to assess cardiac functions and then T2* MRI can be done to assess cardiac iron concentration in selected cases. A fully recovered case of iron-related heart failure is not a contraindication to transplant provided that the patient has received adequate iron chelation therapy.

Endocrine function: Fasting blood glucose levels, thyroid function tests, and growth-hormone-releasing hormone (GHRH) stimulation tests can be performed in children with age more than 10 years to rule out iron-related damage to endocrine organs. These evaluations do not impact transplant outcomes or procedures but can be very useful for long-term post-transplant follow up care.

Fertility assessment: Post pubertal males and females should be encouraged for sperm banking and ovarian tissue preservation respectively as temporary or permanent hypogonadism is common following allogeneic HSCT.

4. Donor selection

The ideal donor for HSCT is HLA-matched/HLA-identical sibling i.e. a sibling who shares the same HLA haplotype at six of six (or eight of eight) HLA loci (HLA—A, B and DR or HLA—A, B, C and DR respectively). The probability of availability of such a donor is 25% and that of a donor without thalassemia major is 18.5%.

5. Evolution of strategies of HSCT in Thalassemia

Over the last 20 years, the probability of thalassemia-free survival in MSD-HSCT has improved from 73% to 80–90% [19]. A survey of 1061 patients who underwent MSD-HSCT in the last 10 years, conducted by EBMT showed two years of overall survival (OS) as 91% [20]. The factors which helped in getting better survival were improved HLA typing, better maintenance of asepsis, more effective prophylaxis and treatment of various infections with the availability of broad-spectrum higher antibiotics, better prophylaxis of graft vs. host disease (GVHD), improved iron chelation before transplant and evolved strategies to deal with Pesaro class 3 transplant. Table 1, highlights the recent evidence from MSD-HSCT in thalassaemic children.

Authors	No. of patients	Patient cohort/overall survival Pesaro risk category	Thalassemia free survival	Treatment related mortality	Comments
Galambrun et al.	108	Children all categories 15 years of risk 86.8%	15 years	15 years	96 sibling donor
			69.40%	12%	Regimen: Bu-Cy ± ATG
Li et al.	82	Children all risk categories 3 years	3 years	3 years	52 MUD, 30 sibling
		91%	87%	8%	Regimen Bu-Cy-Thiotepa, Fludarabine
Bernardo et al.	60	Low: 275 years	5 years	7%	20 sibling donor, 40 MUD
		Intermediate: 17, high: 4 93%	84%		Regimen Treosulfan
		Adults: 12			Thiotepa-Fludarabine
Sabloff et al.	179	Low: 2% 5 years	5 years	Intermediate risk	Bu-Cy + ATG in 77
		Intermediate: Intermediate risk	Intermediate risk	May-75	Bu-Cy in 102
		42% 91%	88%	High risk
		High: 36% High risk: 64%	High risk: 62%	23/64
Irfan et al.	56	Children 5 years	5 years	100 days:	29 BM, 27 PBSCs
		Low: 20 BM: 73%	BM: 67%	Oct-56	Lower risks: Bu-Cy
		Intermediate: 20 PBSCs: 65%	PBSCs: 55%		High risk: Hydroxyurea-
		High: 16			Azathioprine-Fludarabune-
					Bu-Cy
Locatelli et al.	259	Median age 8 years (range 1–24) 6 years	6 years	4%	Multicentric retrospective
		Low: 86 95%	86%		Registry study.
		Intermediate: 122			Regimens: Bu-Cy,
		High: 51			Bu-Cy_Fludarabine, Bu-Cy-
					Thiotepa ±ATG.
Mathews et al.	50	High Risk: 48%	79%	12%	Treo based conditioning with PBSC graft in 74%
Anurathapan et al.	18	NA	89%	5%	Conditioning regimen with Flu and Iv Bu, Preconditioning; Immunosuppression with Fludrabine and dexamethasone for one months.
Gaziev et al.	68	6 low risk 3 years	3 years	100 days	Intravenous Busulfan
		23 intermediate risk 91%	87%	3%	Based regimen
		39 high risk

Table 1.

Recent results of matched sibling donor transplant in children with thalassemia major [16].

OS, overall survival; TFS, thalassemia free survival; TRM, transplant related survival; NR, not reported; Bu, busulfan; Cy, cyclophosphamide; ATG, antithymocyte globulin; BM, bone marrow; PBSC, peripheral blood stem cells; CB, cord blood.

Different centres have come up with strategies for improving outcomes in Pesaro Class 3 patients as can be figured out in Table 1.

1. Hyper transfusion along with effective iron chelation to keep hemoglobin more than 13 g/dl and keep ferritin less than 2000 ng/ml.

2. Reducing the dose of cyclophosphamide (≤160 mg/kg), and adding fludarabine in the conditioning regimen to decrease treatment-related mortality (reduced intensity conditioning; RIC regimens).

3. Adding anti thymocyte globulin (ATG)/total lymphocyte irradiation (TLI)/total body irradiation (TBI) helps in achieving similar survival rates with RIC when compared with myeloablative conditioning regimens (MAC).

4. Pretransplant additional therapies with immunosuppression with azathioprine (Aza) and suppression of erythropoiesis with hydroxyurea (Hu). Sodani et al. [19] reported Protocol 26 which combines RIC regimens with Aza and Hu improving survival to 93% and decreasing graft rejection to 6%

5. Using intravenous (i.v.) busulfan

6. Targeted i.v. busulfan

7. New drugs like treosulfan, thiotepa, and fludarabine (TTF regimen or adding thiotepa individually to the conditioning regimen), as well as intensive pretransplant transfusion-chelation regimens.

8. Gaziev et al. [20] reported Modified Protocol 26. It was similar to that of Sodani et al. but with a higher dose of Fludarabine (150 mg/kg) and the addition of thiotepa (10 mg/kg). This was used in MSD-HSCT with results of 92% OS and no graft rejection.

9. Anurathapan et al. [21] reported a novel reduced toxicity conditioning regimen (RTC) regimen involving sequential administration of Hu (to suppress erythropoiesis) followed by two cycles of Pre transplant immunosuppression (PTIS) including Fludarabine and dexamethasone (to suppress recipient T cells) followed by RTC with Fludarabine, Busulfan and Anti thymocyte globulin along with administration of a relatively high number of hematopoietic progenitor cells (>5 × 106 CD34+ cells/kg of recipient weight). This regimen was used in MSD and unrelated donors and resulted in overall survival of 94% in both groups (Figure 1).

Figure 1.

Depiction of the transplant program with first pharmacological PTIS, followed by RTC with ATG, Flu and IV Bu. For the haplo-identical donors, we used post-Cy-based GvHD prophylaxis and delayed-calcineurin inhibitor-/sirolimus therapy, and short-course mycophenolate mofetil, the latter two starting on day SCT +5. Please see Patients and Methods section for details.

6. Alternative donors

HLA matched sibling as an option for the donor is available only in a quarter of cases. Because of its curative potential, the spectrum of the donor has been extended to alternative donors also. The experience of various types of the donor is as followed:

7. Matched unrelated donor (MUD) transplant

MUD transplant has been reported to be an acceptable strategy for the cure of thalassemia as an alternative option for MSD-HSCT if matched sibling donor is not available. An Italian bone marrow transplant group conducted a study in 2005 regarding MUD transplant using molecular typing in 68 patients with a median age of 15 years. The thalassemia-free survival for 30 patients in Pesaro classes 1 and 2 at a median follow-up of 3.4 years was 97%. However, for the other 38 class 3 patients, the survival was 65% [22]. Experience from several centres suggested that survival can be improved if there is high-resolution molecular typing at both HLA class I and II loci (HLA—A, B, C, DR B1 and DQB1). A recent study has demonstrated that the risk of thalassemia recurrence after unrelated bone marrow transplantation is associated with the presence of nonpermissive HLA-DPB1 mismatches in the host-versus-graft direction [23]. A novel conditioning regimen (WZ-14-TM protocol) based on reduced dose cyclophosphamide, i.v. Busulfan, fludarabine and addition of ATG, used by Chinese group Lan Sun et al. in 48 children of 2–11 years of age with beta-thalassemia major who underwent MUD peripheral blood transplant reported OS and TFS of 100% [24]. In 2021, Kharya et al. reported their experience of using modified PTIS protocol (Apollo Protocol) in 3 thalassemia patients who underwent MUD transplants. Along with Hu, and Aza, they gave 2 cycles of modified PTIS involving decreased cumulative dose of fludarabine as compared to that of Anurathapan et al. along with the addition of cyclophosphamide followed by augmented John Hopkins conditioning and subsequently Post-transplant cyclophosphamide (PtCy). At a median follow-up of 307.5 days, all patients were alive and disease free [23]. The limitations with MUD transplants are limited experience with lesser studies across the globe and the limited number of registries available for searching for matched donors.

8. Mismatched related donor (MMRD)/haploidentical transplant (haploidentical-HSCT)

MMRD/haploidentical transplants are considered experimental strategies to be taken as therapy for Thalassemia. Reports from such transplants are limited but with novel strategies, the outcomes are improving over the years. In 2018, Gaziev et al. reported their experience of using T cell receptor alpha-beta+/CD 19+ depleted grafts (Figure 2) for haplo-HSCT in 14 patients. At the median follow-up of 3.9 years, the five years probability of overall survival was 84%. The incidence of graft failure was 14%. Anurathapan et al. [21] used their Ric protocol (flu-i.v. blu) with PTIS along with ATG, PtCy, tacrolimus and mycophenolate mofetil (MMF) in 83 patients who underwent haplo HSCT. Six patients developed severe acute Graft vs. host disease (GVHD). Projected OS at 3 years was 96%. Experience with haplo-HSCT is very limited and more trials are required in this area.

Figure 2.

T cell depletion.

9. Stem cell source

Stem cells for HSCT can be obtained from bone marrow, peripheral blood and cord blood. In the majority, all the transplant centres across the world use bone marrow as a stem cell source as it is associated with a lesser incidence of GVHD (especially chronic) as compared to peripheral blood because of the high concentration of T lymphocytes in the latter. Overall survival has been better with bone marrow stem cells in some studies. The incidence of graft failure has been similar to all the sources 2014 expert panel guidelines recommend bone marrow as a source of stem cells over peripheral blood [16].

Ghavamzadeh et al. in a 2007 study reported chronic GVHD in 19% of bone marrow HSCT and 48% of peripheral blood HSCT involving 183 children who underwent MSD HSCT [25]. Similar results were seen in a 2010 study which involved 52 children with thalassemia belonging to Pesaro Class 3. They underwent MSD-HSCT. The chronic GVHD was 40% in the group with peripheral blood stem cell transplant and 16% in that with bone marrow stem cell transplant [26]. Cord blood (CB) as a source of stem cells has been tried to extend the donor pool. Also, the rates of acute and chronic GVHD are theoretically less with cord blood. However, the survival rates were similar between cord blood and bone marrow [27]. With unrelated cord blood, high rates of graft failure and delayed hematopoietic recovery were the major concerns [28]. This limitation might be mitigated by using ≥1 CB donor unit or by giving CB together with T cell-depleted HLA-haploidentical CD34 with cells. Current recommendations for CB transplant suggest using units containing at least 3.5 × 10⁷ total nucleated cell/kg recipient body weight before cryopreservation, and with less than 2 HLA disparities.

10. Conditioning regimen

The conditioning regimen for HSCT requires two conditions to be fulfilled. First, ablation of expanded erythropoietic marrow and second immunosuppression of the host for effective engraftment of graft cells. The first condition is fulfilled by Busulfan and its derivative. However, for the second condition busulfan is not that effective and hence cyclophosphamide is incorporated into the backbone as an effective immunosuppressive agent. The myeloablative conditioning derived from this principle was busulfan at the dose of 14–16 mg/kg and cyclophosphamide at the dose of 200 mg/kg. This conditioning regimen has evolved over the years to improve graft survival and overall survival with a lesser incidence of treatment-related toxicities and mortalities. With Busulfan the idea evolved from using it intravenously for effective bioavailability then infusing it with therapeutic drug monitoring [29], then using its derivative treosulfan as in Thiotepa-Treosulfan-Fludarabine (TTF) regimen [30, 31, 32]. With cyclophosphamide, the idea evolved from decreasing its dose from 200 mg/kg to 160 mg/kg [33], which improved survival but increased the graft rejection rate also followed by the addition of fludarabine (RIC regimen) as another immunosuppressive agent followed by the use of pretransplant immunosuppression [21] (Protocol 26, as described above) followed by addition of thiotepa and increasing the dose of fludarabine (modified Protocol 26, as described above), a type of Reduced toxicity Myeloblative (RTM) regimen [34]. As mentioned above Anurathapan et al. [24] and Kharya et al. [23] came up with the novel idea of Pretransplant Immunosuppression and reported optimal survival rates.

11. Graft versus host disease prophylaxis

An international expert panel in 2014 recommended the use of cyclosporine and methotrexate (on days +1, +3, +6, +11 post-transplant) as standard of prophylaxis for GVHD. Cyclosporine is continued for one year [16]. Anti-thymocyte globulin (ATG) has been shown to improve the GVHD rates in MSD, MUD and Haplo-HSCT [35, 36]. It is recommended for use in alternative donor transplants and when using peripheral blood as the source of stem cells. For haploidentical transplants post-transplant cyclophosphamide (PtCy) has also been incorporated for GVHD prophylaxis to get better results [37, 38, 39].

12. Mixed chimerism

Mixed chimerism is defined by the presence of >5% recipient cells at any time post-transplant. The severity is graded as levels 1, 2, 3 depending on the percentage of recipient cells as <10%, 10–25%, and >25% respectively. Andreani et al. [39] showed that engraftment with day +60 chimerism more than 90%, also called bulk engraftment is required for achieving stable complete chimerism or mixed chimerism. Aby Abraham et al. [35] reported the management of mixed chimerism. Initially, they tapered immunosuppression (tapering of dose of cyclosporine by 30% every two weeks) until there was stable mixed chimerism or complete donor chimerism. If there was progressive loss of donor chimerism leading to level 2 or level 3 chimerism on two consecutive occasions despite the tapering of immunosuppression and absence of GVHD, Donor Lymphocyte Infusion (DLI) was given. In their study, 80% of the patients with level II chimerism responded to DLI and 31.2% of those with level III chimerism showed the response. 40% of patients achieved stable mixed chimerism or complete donor chimerism with DLI.

13. Post-transplant management

Post-transplant management includes monitoring of engraftment and chimerism. It also aims at infection prophylaxis, prevention of GVHD, hematopoietic support and management of iron overload post-transplant. Patients are kept at antibacterial, antiviral and antifungal prophylaxis with special attention to protection against Cytomegalovirus (CMV) and Pneumocystis Carini. Transfusion support is given as per needs. GVHD prophylaxis is given as discussed above. Patients should be monitored for organ functions, growth and gonadal functions also. Regarding iron overload, the aim is to keep serum ferritin <2000 ng/ml and liver iron <7.5 mg/g of dry weight. To achieve this a phlebotomy of 5–6 ml/kg every two weeks or four weeks is done until patients are on immunosuppression [40]. After a patient is free of immunosuppression and has stable engraftment, the patient can be kept on iron chelators according to the dosing schedule. Deferiprone should be used with caution as it may cause agranulocytosis.